Medicament comprising as active ingredient N6 substituted adenosine

ABSTRACT

New Medicament comprising as active ingredient a N 6  -substituted adenosine of formula I ##STR1## wherein R 1 , R 2  and R 3  are each independently hydrogen or acyl R 4  -CO- in which R 4  is alkyl, aryl, heteroaryl or aralkyl.

This is a continuation of Ser. No. 807,817 filed Dec. 11, 1985, nowabandoned.

BACKGROUND OF THE INVENTION

The present invention relates to a new medicament, comprising as activeingredient N⁶ substituted adenosine, of formula I ##STR2## in which R₁,R₂ and R₃ are each independently hydrogen or acyl R₄ --CO-- wherein R₄is alkyl, aryl, heteroaryl or aralkyl.

SUMMARY OF THE INVENTION

N⁶ substituted adenosines are already known by U.S. No. 4,340,730 forthe general treatment of hypertension, but the new medicament isdistinguished therefrom by the extent of its field of activity, thepower and duration of its actions.

The method of synthesizing the hydrochlorate of the new compound,designated hereafter Agr 529, will now be described.

A mixture of 4 g (13.2 mmoles) of chloro-6 purine riboside, 3.6 g (26.4mmoles) of amide γ-amino butyric hydrochlorate and 4 g (39.6 mmoles) oftriethylamine are heated at reflux for ten hours in 50 ml of anethanol-water mixture (8:2).

The solution is left to cool, which causes the formation of aprecipitate which is filtered. This precipitate is recrystallized afirst time in water, then in absolute ethanol. 3.5 g of a whiteflocculent precipitate is obtained, i.e. 9.9 mmoles (75%) C.C.M.:Rf=0.66

    ______________________________________                                        Cellulose F plaque      0.1 mm                                                Migration solvent NH.sub.3                                                                            10%                                                   H.sub.2 O               20%                                                   Isopropanol             70%                                                   ______________________________________                                    

Melding point: 215° C.

These 9.9. mmoles of N₆ -(carboxamido-3 propyl) adenosine are dissolvedin 400 ml of hot isopropanol to which 10 mmoles of concentrated 35% HCL,i.e. 0.36 g, are added drop by drop. It is left to cool and the expectedhydrochlorate precipitates. The precipitate is filtered which gives 3 g(78%) of white powder. Melting point: 170° C. Analysis:

    ______________________________________                                        calculated:                                                                             C: 43.5;     H: 5.45; N: 21.67                                      found:    C: 43.20;    H: 5.60; N: 21.30                                      ______________________________________                                    

RMN spectrum: confirms the structure of the product.

A complete pharmacological evaluation is given of the basis medicament,called thereafter Agr 529, comprising an active ingredient--the compoundof formula I, wherein, R₁, R₂ and R₃ each are hydrogen.

The toxicity of Agr 529 was tested on rats, using 7 male rats of 250-300grams, the Agr 529 being administered intraperitoneally. The result ofthe test is given in the table below, given the percentage of mortalityas a function of time.

    ______________________________________                                        TOXICITY OF AGR 529 ON RATS                                                           % Mor-   % Mor-   % Mor- % Mor- % Mor-                                Doses of                                                                              tality at                                                                              tality at                                                                              tality at                                                                            tality at                                                                            tality at                             Agr 529 1 hour   2 hours  6 hours                                                                              24 hours                                                                             7 days                                ______________________________________                                        500 mg/kg                                                                             0%       0%       0%     100%   /                                     350 mg/kg                                                                             0%       0%       0%     100%   /                                     300 mg/kg                                                                             0%       0%       0%      0%    100%                                                                          (48 h)                                250 mg/kg                                                                             0%       0%       0%      0%     0%                                   ______________________________________                                    

It can be seen that for doses less than or equal to 250 mg/kg themortality is zero at 7 days and more.

The pharmacological doses are situated between 0.25 and 50 mg/kg.

ANTI-INFLAMMATORY ACTION

This action was tested on the oedema caused by kaolin in male rats,Spraque-Dawley, of an average weight of 350 g. The animals were dividedat random into four groups, a reference group which receivedintraperitoneally (ip) physiological serum, and three treated groupscalled TESTS 1, TESTS 2, TESTS 3 which received (ip) Agr 529,respectively 5 mg/kg, 10 mg/kg and 50 mg/kg. Then thirty minutes afterthe treatment all the animals received under the plantar 0.1 ml of a 10%kaolin suspension. The volume of the paw was measured by means of aplethysometer (Hugo Basile 7150) one hour before the oedema and one,two, four and five hours afterwards.

The results given in Table 1, (see page 4) show that at the dose of 50mg/kg, the Agr 529 provides 100% inhibition at the fourth hour and hasan anti-inflammatory action which is already considerable for 5 and 10mg/kg.

ANALGESIC ACTION

This action was tested on male mice of 30 grams, the Agr 529 beingadministered intraperitoneally and the test used being that of theheating plate.

The apparatus used was an analgesimeter with electric heating plateSOCREL (Apelex). The temperature of the plate was stabilized at 56° C.The response time was noted corresponding to the time required by theanimal for licking its front or rear paws. For each animal, the averageresponse time corresponds to the average of three consecutivemeasurements. The test was carried out at time 0 then 10 minutes afterthe injection of the drug then 30 minutes afterwards.

The result are give in tables 2 below and 3 (v. pages 5, 6) which showthe important analgesic action of the molecule.

                                      TABLE 1                                     __________________________________________________________________________    Evolution of the volume of the paw expressed as a percentage of the           initial volume.                                                                            Oedema                                                           Treatment    t = 0                                                                              1 hour                                                                              2 hours 4 hours                                                                             5 hours                                 __________________________________________________________________________    References                                                                          Isotonic                                                                             Kaolin                                                                             15.3 ± 2.25                                                                      17.35 ± 2.4                                                                        20.9 ± 2.8                                                                       21.1 ± 5.7                                 aqueous salt              (6)                                                 solution 9%                                                             TESTS 1                                                                             Agr 529                                                                              Kaolin                                                                             8.1 ± 2.3                                                                        11.0 ± 3.3                                                                         9.6 ± 3.3                                                                        12.8 ± 1.6                                 5 mg/kg                   (5)                                           TESTS 2                                                                             Agr 529                                                                              Kaolin                                                                             10.1 ± 1.3                                                                       9.9 ± 1.3                                                                          8.4 ± 1.1                                                                         7.1 ± 1.55                                10 mg/kg                  (5)                                           TESTS 3                                                                             Agr 529                                                                              Kaolin                                                                             1.3 ± 2.4                                                                        -0.15 ± 3.0                                                                        1.3 ± 2.3                                                                        --                                            50 mg/kg                                                                __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________                            Mean time of re-                                                                         Mean response                                      Nature & Doses                                                                        Mean initial                                                                          sponse to the test                                                                       time to the test                                   of the  time of response                                                                      10 min after injec-                                                                      30 mins after injec-                       ANALGESIC                                                                             product test-                                                                         to the test t.sub.o                                                                   tion of the product                                                                      tion of the product                        TEST    ed.     in seconds                                                                            considered t.sub.10 in secs.                                                             considered t.sub.30 in                     __________________________________________________________________________                                       secs.                                      Heating Isotonic                                                                              4.2 ∓ 0.3                                                                          4.9 ∓ 0.4                                                                             4.9 ∓ 0.4                               Plate   aqueous salt                                                          56°                                                                            solution 9%                                                                   iP                                                                    Heating AGR 529 3.4 ∓ 0.5                                                                          18.4 ∓ 2.4                                                                            15.4 ∓ 4.9                              Plate   10 mg/kg                                                              56°                                                                            iP                                                                    Heating AGR 529 3.3 ∓ 0.5                                                                           8.3 ∓ 11.5                                                                           9.2 ∓ 1.9                               Plate   5 mg/kg                                                               56°                                                                            iP                                                                    __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    Study of the analgesic effect consecutive to the intraperitoneal              administration                                                                of Agr 529 at the dose of 1 mg/kg                                                                             Mean time of response to the test                       Nature & Dose                                                                         Mean initial time of response                                                               10 mins after injection of the                          of the product                                                                        to the test in seconds                                                                      product in seconds                            Analgesic Test                                                                          Tested  t = 0         t = 10                                        __________________________________________________________________________    Heating plate 56° C.Heating plate 56° C.                                  n = 8Isontonicaqueous saltsolution 9% i.p.n = 8Agr 5291 mg/kg                 i.p.                                                                                   ##STR3##                                                   __________________________________________________________________________     The comparison of the batches was made by the Student test with: NS: non      significant: and *P < 0.05                                               

THE ACTION ON LOCOMOTOR ACTIVITY

It was tested on mice using an electronic automatic counting devicewhich records separately the displacements and straightening movementsof two batches of five male mice, every five minutes and for an hour.

The evolution of the motor activity is compared with that of a referencebatch in which the animals received isotonic aqueous salt solution underthe same conditions as the batch subjected to the action of the Agr 529intraperitoneally.

It was discovered that from a dose of 1 mg/kg the Agr 529 led to areduction of 75% of activity after one hour.

The molecule of the invention has a highly hypotensive oranti-hypertensive action, as is proved more particularly by the factthat, injected by intraveneous perfusion in rats of 25 to 50 mg/kg, theAgr 529 caused a drop of tension of 40 mm of Hg. Intraperitoneally, adose of 10 mg/kg, it potentializes the drop of tension due tophenobarbital injected intraperitoneally in doses of 12 or 30 mg/kg.

The interest of Agr 529 in anaesthesia is demonstrated by the fact thatin rats, for a dose of pentobarbital (PB) inducing anaesthesic sleep (30mg/kg, i.p.), the administration 30 minutes before of 10 mg/kg of Agr529 increases the duration of the narcosis by 119%.

For an i.p. dose of PB of 12 mg/kg, which does not cause the animal tolose its recovery reflex, the injection (i.p.) thirty minutes before of10 mg/kg of Agr 529 causes a narcosis comparable to that of theanaesthetic doses of PB (30 mg/kg).

From the pharmacological study a large number of pathological disordersmay therefore be proposed as therapeutic indications for use of themolecule of the invention.

The anti-inflammatory, analgesic, tranquilizing, anti-hypertensive ananaesthesic effects have been more particularly mentioned.

As mentioned above, the compound of the invention is distinguished fromknown N⁶ substituted adenosines more especially by the power of itsaction.

Thus it is that if we compare the action on the locomotor activity ofAgr 529 and of the compound of formula (X) ##STR4## it can be seen thatin order to obtain a reduction, after one hour, of the locomotoractivity of 75%, an i.p. injection must be used of

14 mg/kg of compound X

and 1 mg/kg of Agr 529, which in this test seems therefore 14 times morepowerful.

Similarly, the analgesic action of Agr 529 of compound X and anotherknown N⁶ substituted adenosine of formula Y was compared (by the heatingplate test) ##STR5##

The results of the comparative test are given in the following tablewhere the percentages of increase in the response time are given 10minutes and 30 minutes after injection of the drug, with respect to theresponse time at time 0.

    ______________________________________                                                       10 Minutes                                                                              30 Minutes                                           ______________________________________                                        X (70 mg/kg)     +306.25%    +342.85%                                         Y (73 mg/kg)     +135%       +230%                                            Agr 529 (10 mg/kg)                                                                             +440%       +353%                                            ______________________________________                                    

It can be seen that Agr 529 has a considerably higher analgesic actionthan that of compounds X and Y and in particular than that of compoundY.

Besides the intraperitoneal form of administration, the use ofintravenous, intramuscular and oral methods will be proposed clinically.

The intravenous doses in rabbits extends between 1 and 75 mg.kg withdefinitive survival. For testing on human beings, doses of 0.5 to 10mg/kg may be considered for intravenous introduction.

For intramuscular introduction in human beings, an intermediate dose maybe considered to be useful.

Orally (intubation) in animals, doses ten times greater than the dosesused intraperitoneally are required. It may be considered that in humanbeings doses of 50 mg/kg will be useful.

For oral administration, it is preferable to use the compounds offormula I wherein R₁ and/or R₂ and/or R₃ are aryl R₄ --Co, R₄ beingalkyl, aryl etheraly or aralkyl.

There are thus obtained, starting from medicament Agr 529, lipophileesters of the primary alcohol functions which are, what are called"prodrugs" which gives to the Agr 529 a good activity by oraladministration.

As an example of such "prodrugs" is cited the product having as activeingredient the compound of formula I wherein R₁, R₂ and R₃ each are CH₃--CH₂ --CO--, i.e. (Carboxamido-3 propyl)-6, (tripropionyl)-2',3',5', β,D ribosyl)-9 purine of structural formula: C₂₃ H₃₂ N₆ O₈.

The above compound is obtained by reacting with the active ingredient ofAgr 529, pyridine together with propionic anhydride.

Preparation of (Carboxamido-3 propylamino)-6, (tripropionyl 2', 3', 5'β, D ribosyl)-9 purine.

In a flask of 100 ml, are introduced 25 ml of pyridine and 25 ml ofpropionic anhydride. 1 g of N₆ -(carboxamido-3-propyl)adenosine areadded and heated with magnetic stirring to 80° C. This temperature ismaintained until complete dissolving of the N₆-(carboxamido-3-propyl)adenosine. The mixture is then dry evaporated.The resultant oil is purified by elution chromatography on silica(solvent=dichloromethane-methanol: 90-10). With chromatography on thinfilm, a pale yellow oil is obtained.

What is claimed is:
 1. A compound of the formula ##STR6## wherein R₁,R₂, and R₃ are the same or different and each is hydrogen or R₄ --CO--wherein R₄ is alkyl, aryl, heteroaryl, or aralkyl.
 2. A compoundaccording to claim 1 wherein R₁, R₂, and R₃ are each hydrogen.
 3. Acompound according to claim 1 wherein R₁, R₂, and R₃ are each CH₃ --CH₂--CO--.
 4. A lipophilic carboxylic acid ester prodrug of one or more ofthe hydroxyl groups of the compound of the formula ##STR7##
 5. A prodrugaccording to claim 4 wherein the prodrug ester is CH₃ --CH₂ --CO--.
 6. Apharmaceutical composition useful for effecting anti-inflammatory,analgesic, tranquilizing, and anti-hypertensive effects in humans andanimals which comprises a therapeutically effective amount of a compoundof the formula ##STR8## wherein R₁, R₂ and R₃ are the same or differentand each is hydrogen or R₄ --CO-- wherein R₄ is alkyl, aryl, heteroarylor aralkyl, in combination with a pharmaceutically acceptable carrier.7. A composition according to claim 6 wherein R₁, R₂ and R₃ are eachhydrogen.
 8. A composition according to claim 6 wherein R₁, R₂ and R₃are each CH₃ --CH₂ --CO.
 9. A method for treating inflammation, pain,and hypertension, and providing a tranquilizing effect, in humans andanimals which comprises administering to a human or animal in needthereof a therapeutically effective amount of a compound of the formula##STR9## wherein R₁, R₂ and R₃ are the same or different and each ishydrogen or R₄ --CO-- wherein R₄ is alkyl, aryl, heteroaryl or aralkyl,in combination with a pharmaceutically acceptable carrier.
 10. A methodaccording to claim 9 wherein R₁, R₂ and R₃ are each hydrogen.
 11. Amethod according to claim 9 wherein R₁, R₂ and R₃ are each CH₃ --CH₂--CO--.